The role of Ad-36 and its E4orf-1 protein in modulating glycemic control

Current treatment strategies for Type 2 Diabetes Mellitus (T2DM) include a range of anti-diabetic drugs, supplemented by lifestyle modifications to reduce dietary fat intake and body fat. However, for their anti-diabetic action, most drugs recruit insulin signaling pathways, which are already impaired in T2DM. Also, compliance and success in achieving sustained improvements in diet or obesity over the long term is marginal. Therefore, an agent that improves diabetes independent of insulin signaling or lifestyle changes may be highly useful. Human adenovirus Ad36 offers such a model. Ad36 improves glycemic control in chow-fed mice or rats and attenuates diabetes and hepatic steatosis in high fat(HF)-fed mice, despite the HF intake and without reducing adiposity. In human adults, natural Ad36 infection predicts better glycemic control and lower hepatic lipid stores. Ex-vivo cell signaling studies suggest that in mice, Ad36 activates Ras-mediated phosphatidyl- inositol 3-kinase (PI3K) pathway (Ras/PI3K) to up-regulate glucose uptake in skeletal muscle and adipose, and suppresses glucose output from the liver. This study determined if the anti-diabetic properties of Ad36 could be creatively harnessed. Objective 1 determined that Ad36 seropositivity was associated with improved glycemic control and lower hepatic lipids in Caucasian, Hispanic, and African American children and adolescents. Objective 2 determined which of the conventional contributors of insulin sensitivity are modulated by Ad36. In vitro, Ad36 increased preadipocyte differentiation, de-novo lipogenesis, and fat oxidation. Ad36 increased the proportion of small adipocytes in mice on a chow diet, whereas in HF-fed mice, Ad36 increased the proportion of large adipocytes. Adipose tissue macrophage infiltration and angiogenesis were not affected by Ad36. Objective 3 determined the E4orf1 protein of Ad36 mediates its anti-hyperglycemic property. E4orf1 is sufficient and necessary to improve glucose uptake. Mirroring the actions of Ad36, in vitro, E4orf1 also up-regulates the Ras/PI3K pathway, and adiponectin –an insulin sensitizing adipokine, and down-regulates inflammatory cytokine expression. E4orf1 increases glucose uptake in, preadipocytes and adipocytes. In hepatocytes, E4orf1 reduces glucose output and the metabolic studies indicate it favors less hepatic lipid storage. Overall, this study offers a broad foundation to further determine the potential of E4orf1 as an anti-diabetic agent

Longitudinal associations between body mass index, physical activity, and healthy dietary behaviors in adults: A parallel latent growth curve modeling approach

Background Physical activity (PA) and healthy dietary behaviors (HDB) are two well-documented lifestyle factors influencing body mass index (BMI). This study examined 7-year longitudinal associations between changes in PA, HDB, and BMI among adults using a parallel latent growth curve modeling (LGCM). Methods We used prospective cohort data collected by a private company (SimplyWell LLC, Omaha, NE, USA) implementing a workplace health screening program. Data from a total of 2,579 adults who provided valid BMI, PA, and HDB information for at least 5 out of 7 follow-up years from the time they entered the program were analyzed. PA and HDB were subjectively measured during an annual online health survey. Height and weight measured during an annual onsite health screening were used to calculate BMI (kgm2). The parallel LGCMs stratified by gender and baseline weight status (normal: BMI30) were fitted to examine the longitudinal associations of changes in PA and HDB with change in BMI over years. Results On average, BMI gradually increased over years, at rates ranging from 0.06 to 0.20 kgm 2year, with larger increases observed among those of normal baseline weight status across genders. The increases in PA and HDB were independently associated with a smaller increase in BMI for obese males (b = -1.70 and -1.98, respectively), and overweight females (b = -1.85 and -2.46, respectively) and obese females (b = -2.78 and -3.08,respectively). However, no significant associations of baseline PA and HDB with changes in BMI were observed. Conclusions Our study suggests that gradual increases in PA and HDB are independently associated with smaller increases in BMI in overweight and obese adults, but not in normal weight individuals. Further study is warranted to address factors that check increases in BMI in normal weight adults

E4orf1: A Novel Ligand That Improves Glucose Disposal in Cell Culture

Reducing dietary fat intake and excess adiposity, the cornerstones of behavioral treatment of insulin resistance(IR), are marginally successful over the long term. Ad36, a human adenovirus, offers a template to improve IR, independent of dietary fat intake or adiposity. Ad36 increases cellular glucose uptake via a Ras-mediated activation of phosphatidyl inositol 3-kinase(PI3K), and improves hyperglycemia in mice, despite a high-fat diet and without reducing adiposity. Ex-vivo studies suggest that Ad36 improves hyperglycemia in mice by increasing glucose uptake by adipose tissue and skeletal muscle, and by reducing hepatic glucose output. It is impractical to use Ad36 for therapeutic action. Instead, we investigated if the E4orf1 protein of Ad36, mediates its anti-hyperglycemic action. Such a candidate protein may offer an attractive template for therapeutic development. Experiment-1 determined that Ad36 ‘requires’ E4orf1 protein to up-regulate cellular glucose uptake. Ad36 significantly increased glucose uptake in 3T3-L1 preadipocytes, which was abrogated by knocking down E4orf1 with siRNA. Experiment-2 identified E4orf1 as ‘sufficient’ to up-regulate glucose uptake. 3T3-L1 cells that inducibly express E4orf1, increased glucose uptake in an induction-dependent manner, compared to null vector control cells. E4orf1 up-regulated PI3K pathway and increased abundance of Ras–the obligatory molecule in Ad36-induced glucose uptake. Experiment-3: Signaling studies of cells transiently transfected with E4orf1 or a null vector, revealed that E4orf1 may activate Ras/PI3K pathway by binding to Drosophila discs-large(Dlg1) protein. E4orf1 activated total Ras and, particularly the H-Ras isoform. By mutating the PDZ domain binding motif(PBM) of E4orf1, Experiment-4 showed that E4orf1 requires its PBM to increase Ras activation or glucose uptake. Experiment-5: In-vitro, a transient transfection by E4orf1 significantly increased glucose uptake in preadipocytes, adipocytes, or myoblasts, and reduced glucose output by hepatocytes. Thus, the highly attractive anti-hyperglycemic effect of Ad36 is mirrored by E4orf1 protein, which may offer a novel ligand to develop anti-hyperglycemic drugs

Effects of A 12 Week Walking Intervention on Exercise Barriers in Obesity Using an Anti-Gravity Treadmill

Exercise barriers in people with obesity, such as low quality of life, physical activity enjoyment, and self-efficacy may contribute to an increased sedentary lifestyle. However, the use of an anti-gravity treadmill that is able to support and elevate weight during exercise to reduce these barriers has not been examined. PURPOSE: To examine how an anti-gravity treadmill during 12- weeks of aerobic exercise effects Physical activity enjoyment (PAE), physical functioning, self-efficacy, and quality of life (QOL) in people with obesity. METHODS: 26 participants (10 male, 16 female) participated in a self-directed 12 week walking program using an anti-gravity treadmill. Participants were randomized into two groups (N=13/group): weighted (W), exercising at 100% of their weight, or unweighted (UW), who self-selected their workout weight. PAE, self-efficacy, QOL, and timed up and go test (TUG) and a 6 minute walk test were administered pre and post. The QOL questionnaire has different subsections that were used for analysis: overall quality of life, physical health, psychological, social, and environmental. Analysis included participants that completed the pre and post visits (N=17): W (N=9) and UW (N=8). RESULTS: Weight and BMI for W (107.7kg, 36.0kg/m2) and UW (109.8kg, 37.7kg/m2) were not significantly different (P\u3e.05). However age was significantly different, W (27.6 years) and UW (36.4 years) (p=.028). Total duration, and total energy expenditure were not significantly different between groups W (617.5 min, 8501.6kcal) and UW (712.2 min, 9209.7kcal), respectively, (P\u3e.05). After adjusting for baseline values, there wasn’t a significant difference between groups in PAE, self-efficacy, TUG, and the 6 minute walk test (p\u3e.05). As a group, UW scored 13.1 points higher than W on overall quality of life (F=6.601, P=.023). Psychological, social, and environmental subscales of QOL were not significantly different when adjusting for pre values (P\u3e.05). However, physical health QOL was significantly different when adjusting for pre values (F=6.761, P=.020). CONCLUSION: This pilot study confirms that using an anti-gravity treadmill with the unweighting feature can significantly increase overall quality of life and physical health quality of life in people with obesity

Effects of A 12 Week Walking Intervention on Exercise Barriers in Obesity Using an Anti-Gravity Treadmill

Exercise barriers in people with obesity, such as low quality of life, physical activity enjoyment, and self-efficacy may contribute to an increased sedentary lifestyle. However, the use of an anti-gravity treadmill that is able to support and elevate weight during exercise to reduce these barriers has not been examined. PURPOSE: To examine how an anti-gravity treadmill during 12- weeks of aerobic exercise effects Physical activity enjoyment (PAE), physical functioning, self-efficacy, and quality of life (QOL) in people with obesity. METHODS: 26 participants (10 male, 16 female) participated in a self-directed 12 week walking program using an anti-gravity treadmill. Participants were randomized into two groups (N=13/group): weighted (W), exercising at 100% of their weight, or unweighted (UW), who self-selected their workout weight. PAE, self-efficacy, QOL, and timed up and go test (TUG) and a 6 minute walk test were administered pre and post. The QOL questionnaire has different subsections that were used for analysis: overall quality of life, physical health, psychological, social, and environmental. Analysis included participants that completed the pre and post visits (N=17): W (N=9) and UW (N=8). RESULTS: Weight and BMI for W (107.7kg, 36.0kg/m2) and UW (109.8kg, 37.7kg/m2) were not significantly different (P\u3e.05). However age was significantly different, W (27.6 years) and UW (36.4 years) (p=.028). Total duration, and total energy expenditure were not significantly different between groups W (617.5 min, 8501.6kcal) and UW (712.2 min, 9209.7kcal), respectively, (P\u3e.05). After adjusting for baseline values, there wasn’t a significant difference between groups in PAE, self-efficacy, TUG, and the 6 minute walk test (p\u3e.05). As a group, UW scored 13.1 points higher than W on overall quality of life (F=6.601, P=.023). Psychological, social, and environmental subscales of QOL were not significantly different when adjusting for pre values (P\u3e.05). However, physical health QOL was significantly different when adjusting for pre values (F=6.761, P=.020). CONCLUSION: This pilot study confirms that using an anti-gravity treadmill with the unweighting feature can significantly increase overall quality of life and physical health quality of life in people with obesity

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin

E4orf1 improves lipid and glucose metabolism in hepatocytes: a template to improve steatosis & hyperglycemia

Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p\u3c0.0001), and apoB secretion 1.5 fold(p\u3c0.003). Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD)

Transient transfection of 3T3-L1/Tet-On clone by pTRE-Tight-Luc and luciferase assay.

<p>3T3-L1-pTet-On clone was transfected with pTRE-Tight-Luc plasmid. After 48 h post transfection, cells were treated with 0, 100, 1,000 ng/mL or 10,000 ng/mL of Dox and 6 h, 12 h and 48 h post treatment, the luciferase activity was analyzed using the luciferase assay system kit.</p

E4orf1 fold expression relative to pTRE.

<p>The pTRE TIGHT-E4orf1 clone was treated with 0 and 1,000 ng/mL Dox and RNA was harvested at either 16, 24 or 48 h post treatment. E4orf1 fold expression was determined relative to un-induced pTRE TIGHT-E4orf1 at 24 h using real time PCR assay.</p

Effect of regulated <i>E4orf1</i> expression on glucose disposal.

<p>3T3-E4 inducible clone and pTRE empty vector clones were induced with 1,000 ng/mL Dox for 24, 48, 72 and 96 h. After 96 h of induction, Dox was removed for 24 h or 48 h and reintroduced for 24 h or 48 h. At all-time points the glucose uptake in E4orf1 groups was significantly greater compared to the respective pTRE groups as determined by student T-Test (p<0.00001). Mean + SD. The difference of glucose uptake between pTRE and E4orf1 groups is presented, which was calculated by subtracting the average of the pTRE group values from the individual biological replicate value for the respective E4orf1 group.</p